Uncovering the biology of chromatin regulators with drug resistance alleles
Freedy, Allyson Maria
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CitationFreedy, Allyson Maria. 2022. Uncovering the biology of chromatin regulators with drug resistance alleles. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.
AbstractFor millennia, small molecules have served as the backbone of humanity’s therapeutic arsenal. Often less well appreciated is the important role that small molecules have played in the discovery of new biological phenomena. The discovery of new biology by small molecules is often made possible by the study of resistance mutations, genetic variants that suppress a small molecule’s action in a cellular context. In recent years, the field of genome editing has facilitated the identification of drug resistance mutations with unprecedented breadth and depth. This new technology has allowed for the identification of small molecule resistance mutation profiles, which have catalyzed the discovery of new molecular mechanisms and therapeutic vulnerabilities across diverse fields of inquiry.
This work seeks to illustrate the role that resistance mutations can play in the discovery of unexpected biological phenomenon. Specifically, I focus upon two projects in which the study of small molecule resistance mutations advanced our understanding of two hematologic malignancies: acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL).
In Chapter 1, I review key examples in which the identification and characterization of resistance mutations have led to the discovery of new biology across a diversity of fields. These examples are grouped by the role that resistance mutations served to illuminate new biology in each context. I also discuss recent advances in genome editing technology and comment on the potential impact that these technologies may have on the identification and characterization of drug resistance mutations moving forward.
In Chapter 2, I discuss our work investigating the biology of AML through the characterization of resistance mutations to small molecule inhibitors of the histone demethylase, LSD1. Prior to our work, it was known that LSD1 was essential for AML survival, but the precise molecular function of LSD1 that was essential for leukemia cell growth was unclear. To clarify the role of LSD1 in leukemia, we profiled resistance mutations to LSD1 inhibitors in AML cell models using our method, CRISPR-suppressor scanning. Through this approach, we identified resistance mutations that both impair drug binding and disrupt LSD1 enzyme activity, suggesting that LSD1 demethylase activity is not required for AML survival. We further demonstrated that drug-mediated disruption of a complex between LSD1 and the transcription factor, GFI1B, is necessary to block AML growth. Through these studies, we revised the mechanism of action of LSD1 inhibitors and clarified the role of LSD1 in AML.
In Chapter 3, I describe how our discovery of drug addiction mutations in the transcriptional repressor PRC2 led to the identification of a repressive methylation ceiling in DLBCL. Recurrent activating mutations in EZH2, the catalytic subunit of PRC2, promote neoplastic growth in follicular and diffuse large B-cell lymphoma. Using a method which we termed CRISPR-addiction scanning, we identified mutations that confer drug addiction to inhibitors of PRC2 in EZH2-mutant lymphoma. We found that these drug addiction mutations elevate PRC2 methyltransferase activity such that upon drug discontinuation there is overspreading of H3K27me3, oncogene silencing and ultimately cell death. This finding revealed that EZH2-mutant lymphomas live in a ‘Goldilocks’ epigenetic state bounded by a previously unknown repressive methylation ceiling, suggesting that PRC2 activation may represent a new cancer vulnerability in this context.
Taken together, these results showcase how the study of drug resistance mutations can pinpoint small molecule mechanism of action, uncover protein allostery and reveal new therapeutic opportunities. These findings advance our understandings of these two cancers and suggest that resistance mutation profiling is warranted more broadly in other cancer contexts to uncover similar biological insights.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37373607
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